CN110669792A - Genetically modified mesenchymal stem cell, preparation method, application and cell therapy product - Google Patents
Genetically modified mesenchymal stem cell, preparation method, application and cell therapy product Download PDFInfo
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Abstract
The invention provides a gene modified mesenchymal stem cell, a preparation method, an application and a cell therapy product, and relates to the technical field of biotechnology, gene therapy and drug development. The method has the advantages of simple process, convenient operation, batch production, realization without expensive instruments and effective cost saving. Meanwhile, the expression of the gene modified mesenchymal stem cell modified gene prepared by the method has no obvious difference with that of a freshly prepared cell, and the stable expression of the modified gene can be ensured on the basis of saving time and cost, so that the activity and the curative effect of the gene modified mesenchymal stem cell are further ensured.
Description
Technical Field
The invention relates to the technical fields of biotechnology, gene therapy and drug development, in particular to a gene modified mesenchymal stem cell, a preparation method, application and a cell therapy product.
Background
Stem cells have the potential for self-renewal and directed differentiation, and have been shown to promote the repair of various tissue injuries, such as radiation lung injury, chronic liver injury, ulcerative colitis, and the like. Mesenchymal Stem Cells (MSCs) are important adult stem cells, have the advantages of high self-renewal capacity, multidirectional differentiation potential, convenient material acquisition and the like, are attracted by attention, have the advantage of immune exemption and are ideal cell types for stem cell therapy. MSCs have significant efficacy in basic and clinical studies of a variety of diseases, including myocardial infarction, nervous system, diabetes, cartilage and bone injury, crohn's disease, graft-versus-host disease (GVHD), and the like. However, in the damaged part of the disease, there are often significant pathophysiological environments such as ischemia, hypoxia, inflammation, oxidative stress, etc., which can significantly reduce the survival and efficacy of transplanted MSCs, thereby affecting the therapeutic effect of MSCs and being difficult to achieve expectations. Therefore, the enhanced MSCs are obtained through gene modification, survival and implantation of the MSCs at the damaged parts are promoted, and the method has important significance for expanding and enhancing the application and curative effect of the MSCs.
The role and mechanism of MSCs in tissue and organ injury repair mainly include the following aspects: (1) directional differentiation: MSCs can home to damaged parts, differentiate into specific cell types (such as intestinal epithelial cell and the like) under the action of stimulating factors, repair damaged tissues and restore tissue functions; (2) immune and inflammatory modulation: MSCs can inhibit various immune cell activities (such as DC, T, B lymphocytes, NK cells and the like) and inhibit secretion of various inflammatory factors (such as IFN-gamma, IL-12, TNF-alpha and the like); meanwhile, the compound can also inhibit the functions of macrophages and T lymphocytes by secreting various anti-inflammatory factors such as IL-4, IL-10 and the like so as to resist inflammatory reaction caused by IL-1, IL-6, IL-8 and the like; (3) promoting the regeneration of tissue cells: by releasing various growth factors, such as vascular endothelial cell growth factor (VEGF), insulin-like growth factor (IGF-1), HGF and the like, the effects of resisting apoptosis, promoting proliferation, promoting angiogenesis and the like are exerted, and the aim of promoting tissue repair is further fulfilled; (4) and others: MSCs can also inhibit disease progression through a variety of mechanisms, e.g., MSCs can inhibit the progression of tissue and organ fibrosis by inhibiting the deposition of type I and type III collagen and the expression of genes and proteins such as matrix metalloproteinases (MMP-1) and tissue inhibitors of metalloproteinases (TIMP-1).
Although MSCs have a wide range of functions of inflammation regulation, tissue repair and regeneration, they cannot effectively target key molecules or links of disease occurrence and development. Therefore, the clinical effect is often difficult to achieve. The key link of enhancing the local colonization and differentiation of the MSCs in the injury by a gene modification means and inhibiting the occurrence and development of diseases is significant for improving the treatment effect of the MSCs. Currently, the therapeutic studies of genetically modified MSCs are in the beginning and most protocols employ freshly prepared cells. Although freshly prepared cells have the advantages of high cell viability and the like, the freshly prepared cells also have the defects of high preparation cost (mass production is impossible), difficult quality control (limited quality inspection time after cell preparation), poor drug-forming property and the like. The establishment of a preparation method of a cryopreserved high-efficiency gene modified MSCs product has important significance for improving the pharmacy of the MSCs.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide a method for preparing a genetically modified mesenchymal stem cell, which alleviates at least one of the technical problems of the prior art.
The second purpose of the invention is to provide the gene modified mesenchymal stem cell prepared by the preparation method.
The third purpose of the invention is to provide the preparation method or the application of the gene modified mesenchymal stem cells in preparing cell therapy products.
It is a fourth object of the present invention to provide a cell therapy product.
The fifth object of the present invention is to provide the use of the above cell therapy product for the preparation of a product for the treatment of paraquat poisoning.
The invention provides a preparation method of a gene modified mesenchymal stem cell, which comprises the following steps: and modifying the mesenchymal stem cells by using the recombinant adenovirus, and performing frozen storage after 2-4h of modification to obtain the gene modified mesenchymal stem cells.
Further, the modification time is 3 h.
Further, the recombinant adenovirus comprises a defective recombinant adenovirus, preferably comprising ad.hgf, ad.dcn or ad.luc.
Further, the recombinant adenovirus has a multiplicity of infection of 80-150MOI, preferably 100 MOI.
Further, the mesenchymal stem cell comprises a dental pulp mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell or a bone marrow mesenchymal stem cell.
The invention also provides the gene modified mesenchymal stem cell prepared by the preparation method.
The invention also provides the preparation method or the application of the gene modified mesenchymal stem cells in preparing cell therapy products.
Further, the cell therapy product includes a cell therapy drug;
preferably, the cell therapy drug comprises an external cell therapy drug or an injection cell therapy drug.
The invention also provides a cell therapy product comprising the genetically modified mesenchymal stem cell described above;
preferably, the cell therapy product comprises a cell therapy drug;
preferably, the cell therapy drug comprises an external cell therapy drug or an injection cell therapy drug.
The invention also provides application of the cell therapy product in preparing a product for treating paraquat poisoning.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the gene modified mesenchymal stem cell provided by the invention comprises the steps of modifying the mesenchymal stem cell by using the recombinant adenovirus, and performing frozen storage after 2-4h of modification to obtain the gene modified mesenchymal stem cell. The method has the advantages of simple process, convenient operation, batch production, realization without expensive instruments and effective cost saving. The recombinant adenovirus has the advantage of wide host range, can be widely applied to modification of different mesenchymal stem cells, and the mesenchymal stem cells modified by the recombinant adenovirus can effectively improve the treatment effect on key molecules or links of disease occurrence and development. Meanwhile, the preparation method can be implemented after the recombinant adenovirus modifies the mesenchymal stem cells for 2-4h, the quality inspection time is sufficient, and the quality of the genetically modified mesenchymal stem cells can be effectively controlled. Compared with longer modification time such as 24h or 48h and then freezing storage, the expression of the gene modified mesenchymal stem cell modified gene prepared by the method has no obvious difference with that of a freshly prepared cell, and the stable expression of the modified gene can be ensured on the basis of saving time and cost, so that the activity and curative effect of the gene modified mesenchymal stem cell are further ensured.
The gene modified mesenchymal stem cell is prepared by the preparation method provided by the invention, can be used as a stock solution to be stored in liquid nitrogen for a long time, does not influence the expression efficiency of a target gene and the survival in vivo after recovery, and can effectively improve the drug forming property of the gene modified mesenchymal stem cell.
In addition, the inventor of the invention discovers through experiments that the gene modified mesenchymal stem cell prepared by the preparation method provided by the invention can obviously improve the survival rate of mice in the treatment of paraquat poisoning mice compared with the simple mesenchymal stem cell, is expected to expand the clinical application of the gene modified mesenchymal stem cell and improve the treatment level of paraquat poisoning.
Drawings
Fig. 1A is a flow cytometry detection result of a PE-labeled heterogeneous control of dental pulp mesenchymal stem cells according to example 2 of the present invention;
fig. 1B is a flow cytometry detection result of HLA-DR of dental pulp mesenchymal stem cells provided in example 2 of the present invention;
fig. 1C is a flow cytometry detection result of dental pulp mesenchymal stem cell CD105 provided in example 2 of the present invention;
fig. 1D is a flow cytometry detection result of FITC-labeled heterotypic control of dental pulp mesenchymal stem cells provided in embodiment 2 of the present invention;
fig. 1E is a flow cytometry detection result of dental pulp mesenchymal stem cell CD45 provided in example 2 of the present invention;
fig. 1F is a flow cytometry detection result of dental pulp mesenchymal stem cell CD19 provided in example 2 of the present invention;
fig. 1G is a flow cytometry detection result of dental pulp mesenchymal stem cell CD34 provided in example 2 of the present invention;
fig. 1H is a flow cytometry detection result of dental pulp mesenchymal stem cell CD11b provided in example 2 of the present invention;
fig. 1I shows the result of flow cytometry detection of dental pulp mesenchymal stem cell CD73 provided in example 2 of the present invention;
fig. 1J is a flow cytometry detection result of dental pulp mesenchymal stem cell CD90 provided in example 2 of the present invention;
fig. 2 is a result of examining osteogenic (left) differentiation and adipogenic (right) differentiation abilities of dental pulp mesenchymal stem cells provided in example 2 of the present invention;
fig. 3 shows the detection result of HGF expression after preparing the dental pulp mesenchymal stem cells modified with the ad.hgf gene according to the different processes provided in embodiment 4 of the present invention;
FIG. 4A shows the in vivo imaging detection results of nude mice subcutaneously transplanted with Ad-luc gene-modified dental pulp mesenchymal stem cells prepared by cryopreservation 3h after infection according to example 5 of the present invention;
FIG. 4B is a result of in vivo imaging detection of nude mice subcutaneously transplanted with Ad-luc gene-modified dental pulp mesenchymal stem cells prepared by cryopreservation 24h after infection according to example 5 of the present invention;
FIG. 5 shows the result of in vivo imaging detection statistical analysis after nude mice subcutaneously transplanted with Ad-luc gene modified dental pulp mesenchymal stem cells prepared by different processes provided in example 5 of the present invention;
FIG. 6 shows the result of detecting cell phenotype of the genetically modified umbilical cord mesenchymal stem cell provided in example 6 of the present invention;
fig. 7 is a result of measuring osteogenic (left) differentiation and adipogenic (right) differentiation abilities of the genetically modified umbilical cord mesenchymal stem cell provided in example 6 of the present invention;
fig. 8 is a comparison of survival rates of mice treated with the genetically modified dental pulp mesenchymal stem cells according to example 7 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.
Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.
The method for enhancing the mesenchymal stem cells by means of gene modification has important significance for improving the treatment effect of the mesenchymal stem cells, however, the currently adopted mesenchymal stem cell gene modification means generally needs freshly prepared cells, and the problems of high cost and difficult quality control generally exist. Based on this, according to a first aspect of the present invention, there is provided a method for preparing a genetically modified mesenchymal stem cell, the method comprising: and modifying the mesenchymal stem cells by using the recombinant adenovirus, and performing frozen storage after 2-4h of modification to obtain the gene modified mesenchymal stem cells.
The preparation method of the gene modified mesenchymal stem cell provided by the invention has the advantages of simple process, convenient operation, batch production, realization without expensive instruments and effective cost saving. The recombinant adenovirus has the advantage of wide host range, can be widely applied to modification of different mesenchymal stem cells, can be effectively propagated, has higher titer, is more suitable for improving the application effect of the genetically modified mesenchymal stem cells, and can effectively improve the treatment effect on key molecules or links of disease occurrence and development by applying the mesenchymal stem cells modified by the recombinant adenovirus. Meanwhile, the preparation method can be implemented after the recombinant adenovirus modifies the mesenchymal stem cells for 2-4h, the quality inspection time is sufficient, and the quality of the genetically modified mesenchymal stem cells can be effectively controlled. Compared with longer modification time such as 24h or 48h and then freezing storage, the expression of the gene modified mesenchymal stem cell modified gene prepared by the method has no obvious difference with that of a freshly prepared cell, and the stable expression of the modified gene can be ensured on the basis of saving time and cost, so that the activity and curative effect of the gene modified mesenchymal stem cell are further ensured.
It should be noted that, in the present invention, the recombinant adenovirus is derived from a recombinant adenovirus plasmid vector, and the modified recombinant adenovirus is not limited, and all recombinant adenoviruses capable of improving the application effect of the mesenchymal stem cell by modification are within the protection scope of the present invention. The invention uses the recombinant adenovirus to modify the mesenchymal stem cells and has the following advantages: (1) the gene capacity is large: the whole expression frame can be inserted to realize physiological level expression or simultaneously express a plurality of target genes; (2) the gene expression efficiency is high; (3) the recombinant adenovirus is easy to prepare high-titer products and has low cost; (4) the safety is high: does not integrate into the genome.
In order to improve the safety of the genetically modified mesenchymal stem cell, in some preferred embodiments, the recombinant adenovirus comprises a defective recombinant adenovirus, and the defective recombinant adenovirus can avoid long-term large-scale cloning of the virus on the basis of effective modification, so that the use safety of the prepared genetically modified mesenchymal stem cell can be further enhanced.
Preferably, the recombinant adenovirus comprises ad.hgf, ad.dcn or ad.luc.
In some preferred embodiments, the recombinant adenovirus has a multiplicity of infection of 80-150MOI, for example, but not limited to, 80MO, 90MOI, 100MOI, 110MOI, 120MOI, 130MOI, 140MOI or 150MOI, and when the multiplicity of infection of the recombinant adenovirus is within the above range, it is more ensured that the modified gene is stably expressed in the genetically modified mesenchymal stem cell prepared.
Preferably, the recombinant adenovirus has a multiplicity of infection of 100 MOI.
The inventor of the invention further adjusts and optimizes the infection complex number of the recombinant adenovirus, and finds that after the mesenchymal stem cells are subjected to gene modification by adopting the 100MOI recombinant adenovirus, the survival of the cells and the expression of target genes are not significantly influenced by direct transplantation or transplantation after cryopreservation.
In the present invention, the modification time may be, for example, but not limited to, 2h, 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h, 3.8h or 4 h. The mesenchymal stem cells are frozen and preserved after being modified for 2-4h by using the recombinant adenovirus, and have higher expression level of the modified gene and survival in vivo after transplantation after recovery.
Preferably, the time of modification is 3 h.
The inventors of the present invention further found that the expression level of the modified gene and the survival in vivo after transplantation can be further improved by cryopreserving the recombinant adenovirus gene for 3 hours after modification, compared with cryopreserving the recombinant adenovirus gene for a longer time such as 24 hours or 48 hours after modification.
In the present invention, the modified mesenchymal stem cell is not limited, and preferably, a dental pulp mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell, or a bone marrow mesenchymal stem cell may be modified.
According to the second aspect of the invention, the genetically modified mesenchymal stem cell prepared by the preparation method is also provided.
The gene modified mesenchymal stem cell is prepared by the preparation method provided by the invention, can be used as a stock solution to be stored in liquid nitrogen for a long time, does not influence the expression efficiency of a target gene and the survival in vivo after recovery, and can effectively improve the drug forming property of the gene modified mesenchymal stem cell.
According to the third aspect of the invention, the preparation method or the application of the gene modified mesenchymal stem cell in preparing cell therapy products is also provided.
The mesenchymal stem cells have wide functions of inflammation regulation, tissue repair, regeneration and the like, and have obvious curative effects in the basic and clinical research of various diseases.
In some preferred embodiments, the cell therapy product comprises a cell therapy drug.
Preferably, the cell therapy drug comprises an external cell therapy drug or an injection cell therapy drug.
According to a fourth aspect of the present invention, there is also provided a cell therapy product comprising the genetically modified mesenchymal stem cell described above.
The modified gene expression rate and the in vivo survival rate of the genetically modified mesenchymal stem cell provided by the invention are high, and the drug forming property of the genetically modified mesenchymal stem cell can be effectively improved, so that a cell therapy product containing the genetically modified mesenchymal stem cell provided by the invention can effectively treat key molecules or links of disease occurrence and development, and has high activity and good curative effect.
Furthermore, according to a fifth aspect of the present invention, there is provided the use of the cell therapy product described above in the manufacture of a product for the treatment of paraquat poisoning.
The inventor of the invention discovers through experiments that the gene modified mesenchymal stem cell prepared by the preparation method provided by the invention can obviously improve the survival rate of mice in the treatment of paraquat poisoning mice compared with the simple mesenchymal stem cell, is expected to expand the clinical application of the gene modified mesenchymal stem cell and improve the treatment level of paraquat poisoning.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The main reagents and instrument information used in the embodiment of the invention are as follows:
clean bench [ Thermo, 1300 ]; carbon dioxide incubator [ Thermo, 311 ]; inverted microscope [ BM-37XB ]; cell counter [ Countstar-IC1000 ]; low temperature horizontal centrifuge [ Thermo, Sorvall ST 8R ]; biosafety cabinet [ Thermo, 1300 sera a2 ]; flow cytometry [ BD Bioscience, FACS calibur ];
the main reagents are as follows:
| reagent | Brand | Batch number |
| Sodium chloride injection | Four medicines | 1710223102 |
| 0.25% Trypsin | GIBCO | 1836501 |
| 0.4% Trypan blue | GIBCO | 1854966 |
| Serum-free frozen stock solution MSC Freezing | BI | 1823345 |
| Recombinant adenovirus | Company self-made |
Example 1 isolation and culture of dental pulp mesenchymal stem cells
After the donor signs an informed consent, in the medical institution having the donor screening ability who obtains the "medical institution license of medical institution", the medical personnel with abundant experience of holding the doctor or nurse certificate of license are used to aseptically extract the third molar without dental caries, pulpitis and pulp necrosis under anesthesia according to the diagnosis and treatment routine. The freshly extracted teeth are immediately placed into a tooth collecting bottle (filled with 20ml of sterile physiological saline) meeting the requirements, and dental pulp mesenchymal stem cells are separated and cultured within 24 hours. The specific method comprises the following steps: cutting dental crown, collecting dental pulp tissue, repeatedly washing with normal saline, and cutting into 1mm pieces3The left and right small blocks; placing in physiological saline containing 3mg/ml type I collagenase and 4mg/ml Dispase, and performing shake digestion in water bath at 37 ℃ for 40 min; sieving with 70 μm cell sieve, centrifuging at 1000rpm for 10min, and collecting cells; resuspending the cells in a suitable amount of alpha-MEM medium containing 20% fetal bovine serum to give a single cell suspension, inoculating the cells in a 10cm dish, incubating at 37 deg.C and 5% CO2Culturing, and changing the culture solution once in 3-5 days. The growth condition of the cells is observed under an inverted microscope every day, and after 12 to 16 days, the clonally grown cells are digested and collected by 0.25% trypsin to obtain the P0 generation dental pulp mesenchymal stem cells.
Example 2 identification and expansion of dental pulp mesenchymal stem cells
Collection of P0-generation mesenchymal stem cells (dental pulp, umbilical cord, bone marrow, fat) obtained in example 1 at 8000 cells/cm2After culturing for 4 days, the cells were collected by digestion with 0.25% trypsin and passaging was continued. Passage to P5 passage, cell phenotype, Prostaglandin E2(Prostaglandin E2, PGE2), and multiple differentiation (osteogenic and adipogenic differentiation) potential assays were performed.
(1) Immunophenotypic testing
After the mesenchymal stem cells are digested by 0.25 percent trypsin, the cells are labeled by antibodies such as CD73, CD90, CD105, CD11b, CD19, CD34, CD45, HLA-DR and the like marked by FITC or PE and corresponding isotype control antibodies, and detected by a flow cytometer. The detection results are shown in fig. 1A-fig. 1J, the dental pulp mesenchymal stem cells highly express mesenchymal stem cell specific antigens CD73, CD90 and CD105, do not express hematopoietic and immune cell surface markers CD11b, CD19, CD34 and CD45, and hardly express surface markers HLA-DR related to transplant immune rejection. These results suggest that highly pure dental pulp MSCs were successfully obtained by the separation method of example 1.
(2) PGE2 expression
PGE2 is an important cell growth and regulatory factor that plays an important role in the regulation of immune and inflammatory processes mediated by mesenchymal stem cells. P4 dental pulp mesenchymal stem cells according to 8000 cells/cm2Density of (3) inoculation of 75cm2After the flask, 5% CO at 37 ℃2And 4d, collecting supernatant, and detecting the expression of PGE2 by using an ELISA detection kit, wherein the result shows that the expression amount of PGE2 is more than 200 pg/ml.
(3) Multidirectional differentiation potential detection
Osteogenic differentiation: replacing the P5 dental pulp mesenchymal stem cells by 1 × 104Inoculating 24-well culture plate to cells/well, and culturing at 37 deg.C and 5% CO2Culturing in a saturated humidity incubator, adding osteogenic inducing solution after the cells reach about 80% fusion degree, and changing the solution every 4 days. Identification was carried out by alizarin red staining after 21 days of induction.
Adipogenic differentiation: the generation of P5 dental pulp mesenchymal stem cells is performed at 1.5 × 104Inoculating 24-well culture plate to cells/well, and culturing at 37 deg.C and 5% CO2And culturing in an incubator with saturated humidity. After 24 hours, the adipogenic induction solution was added and the solution was changed every 4 days. After 14 days of induction, identification was carried out by staining with oil Red O (oil Red O).
The detection result is shown in fig. 2, which shows that the dental pulp mesenchymal stem cells obtained by separation have osteogenic and adipogenic differentiation potential. In conclusion, the dental pulp mesenchymal stem cells with high quality are successfully separated and amplified in the embodiment 1 of the invention.
Example 3 dental pulp mesenchymal stem cell Gene modification and cryopreservation Process
The generation of P5 dental pulp mesenchymal stem cells is according to 16000 cells/cm2Density of (2) inoculation 175cm2The culture flask of (1), after being placed at 37 ℃ and 5% CO2Culturing in a saturated humidity incubator for 48h, adding 100MOI (IU/cell) recombinant adenovirus vector (Ad. HGF, Ad) when the cells reach 70% -80% fusion degreeDCN and ad.luc), after 3h of co-incubation: (1) digesting and collecting cells by using 0.25% trypsin, and freezing and storing the cells by using a serum-free freezing medium; (2) discarding the culture medium containing the virus solution, washing with normal saline twice, adding the mesenchymal stem cell serum-free culture medium, continuing culturing for 24h, digesting and collecting the cells with 0.25% trypsin, and freezing the cells by adopting a serum-free freezing medium. 3h after infection and 24h after infection, the state of the dental pulp mesenchymal stem cells is good.
Example 4 Effect of cryopreservation Process on survival Rate of dental pulp mesenchymal Stem cells and exogenous Gene expression
And (3) after the dental pulp mesenchymal stem cells modified by the HGF gene are frozen and stored for 2 weeks, recovering the cells, counting and calculating the cell survival rate. The results are shown in table 1, which indicates that recombinant adenovirus-mediated gene modification (HGF and luciferase) has no significant effect on the cell viability rate after cryopreservation of dental pulp mesenchymal stem cells.
TABLE 1 Ad.HGF Gene modified cells viability Rate before and after cryopreservation
Revived cells at 8000 cells/cm26-well plate is inoculated in the density of the seed; fresh cells infected with ad.hgf 3h and 48h were used as controls. Placing it at 37 ℃ and 5% CO2Continuously culturing for 24 hours or 48 hours in an incubator with saturated humidity, and collecting supernatant; centrifuging at 4 deg.C and 3000rpm for 10min, sucking supernatant, and freezing at-80 deg.C.
The concentration of HGF in the supernatant is detected by adopting an ELISA detection kit, the result is shown in figure 3, the group is frozen after 24h of infection, and the HGF expression amount after 24h and 48h of culture is obviously lower than that of the freshly prepared genetically modified cell; however, the HGF expression levels 24h and 48h after culture in the groups frozen 3h after infection were comparable to, and even higher than, those of freshly prepared genetically modified cells.
Example 5 Effect of cryopreservation Process on in vivo survival of genetically modified dental pulp mesenchymal Stem cells
Based on the results of in vitro studies, in vivo transplantation studies were performed using dental pulp mesenchymal stem cells modified with ad.luc genes. Genetically modified dental pulp mesenchymal stem cells (dpsc. luc.f3h) cryopreserved 3h after infection and fresh genetically modified dental pulp mesenchymal stem cells (dpsc. luc.n3h) collected 24h after infection were prepared according to the methods of example 3, respectively, and then nude mice were transplanted subcutaneously using a subcutaneous transplantation method, and the survival time of the dental pulp mesenchymal stem cells was observed using a living body imaging technique. The detection results are shown in fig. 4A, fig. 4B and fig. 5, and it can be seen that at different time points after transplantation, the fluorescence intensity of the dpsc.luc.f3h transplanted group is equivalent to or even higher than dpsc.luc.n3h, which indicates that there is no significant effect on survival of the dental pulp mesenchymal stem cells and expression of the modified gene after 3h cryopreservation after gene modification.
Example 6 Effect of cryopreservation Process on genetically modified umbilical cord mesenchymal Stem cells
(1) Isolated culture of umbilical cord mesenchymal stem cells
After the parturient agreed and signed the informed consent, the umbilical cord was stored in a sterile glass bottle containing a medium containing 300ml of α -MEM, stored at 4 ℃, and the umbilical cord stem cells were isolated and cultured within 24 hours after collection. Repeatedly cleaning with sterile normal saline, cutting into 2cm segments, removing umbilical vein and umbilical artery, and collecting jelly Wharton's jelly; cutting into 5mm pieces3Placing the tissue blocks in a prewetted 10cm culture dish by a dropper, adding a small amount of serum-free culture medium, and culturing at 37 deg.C with 5% CO2Culturing for 48 h; continuing culturing after supplementing the solution, and changing the solution once every 3-4 days; long spindle cells can climb out after 7-10 days; and removing tissue blocks after 10-14 days, and digesting and collecting clonally grown cells by using 0.25% trypsin to obtain the P0 generation umbilical cord mesenchymal stem cells.
(2) Genetic modification of umbilical cord mesenchymal stem cells
According to the dental pulp mesenchymal stem cell gene modification method, 100MOI recombinant adenovirus vector Ad.HGF is adopted to infect umbilical mesenchymal stem cells, after incubation for 3h, 0.25% trypsin is used to digest and collect the cells, and serum-free frozen stock solution is adopted to freeze and store the cells.
(3) Immunophenotypic testing
After being frozen and stored for 14 days, the gene modified umbilical cord mesenchymal stem cells are recoveredAt 16000 cells/cm2Inoculating 6-well plates, culturing for 48h, digesting with 0.25% trypsin, labeling cells with antibodies labeled by FITC or PE, such as CD73, CD90, CD105, CD11b, CD19, CD34, CD45, HLA-DR and the like, and corresponding isotype control antibodies, and detecting by a flow cytometer. The detection results are shown in figure 6, and the results indicate that the phenotype of the umbilical cord mesenchymal stem cells is not obviously influenced by the gene modification and cryopreservation process.
(4) Multidirectional differentiation potential detection
The osteogenic and adipogenic capacity of the genetically modified umbilical cord mesenchymal stem cells was examined according to the method of example 2. The detection result is shown in figure 7, which shows that the gene modification and cryopreservation process has no significant influence on the osteogenic and adipogenic differentiation potential of the umbilical cord mesenchymal stem cells.
Example 7 evaluation of therapeutic Effect of the genetically modified dental pulp mesenchymal Stem cells on paraquat poisoning
After qualified by quarantine, female C57BL/6J mice are divided into layers and randomly according to weight, and the method comprises the following steps: solvent control group, model control group, umbilical cord mesenchymal stem cell group, dental pulp mesenchymal stem cell group (1 × 10)6) HGF gene-modified dental pulp mesenchymal stem cell group (DPSC. HGF) (1 × 106) Dcn (1 × 10) and decorin-modified dental pulp mesenchymal stem cell group (dpsc6). Before molding, all animals are fasted overnight (more than 12 hours without water), sterilized water for injection is given to a solvent control group by gavage, other groups are molded by gavage with 150mg/kg of PQ, and the animals start to be fed with the feed normally about 2 hours after molding. Cells were given at D1 (post PQ molding), D3 days, and all surviving animals were euthanized at day 28. Referring to fig. 8, the survival curves of animals suggest that both dental pulp mesenchymal stem cells and umbilical cord mesenchymal stem cells can improve the survival rate of mice to a certain extent, and the genetically modified dental pulp mesenchymal stem cells prepared in example 3 have more significant advantages.
In a word, after gene modification is carried out on the 100MOI recombinant adenovirus vector for 3 hours, the cryopreservation has no obvious influence on the activity of the mesenchymal stem cells including the dental pulp mesenchymal stem cells and the expression of the modified genes, and the treatment effect of the gene modified cells can be guaranteed.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A preparation method of a genetically modified mesenchymal stem cell, which comprises the following steps: and modifying the mesenchymal stem cells by using the recombinant adenovirus, and performing frozen storage after 2-4h of modification to obtain the gene modified mesenchymal stem cells.
2. The method according to claim 1, wherein the modification time is 3 hours.
3. The method of claim 1, wherein the recombinant adenovirus comprises a defective recombinant adenovirus, preferably comprising ad.hgf, ad.dcn, or ad.luc.
4. The method of claim 1, wherein the recombinant adenovirus has a multiplicity of infection of 80 to 150MOI, preferably 100 MOI.
5. The method of any one of claims 1-4, wherein the mesenchymal stem cell comprises a dental pulp mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell, or a bone marrow mesenchymal stem cell.
6. Genetically modified mesenchymal stem cells prepared by the method of any one of claims 1 to 5.
7. Use of the preparation method of any one of claims 1 to 5 or the genetically modified mesenchymal stem cell of claim 6 for the preparation of a cell therapy product.
8. The use of claim 7, wherein the cell therapy product comprises a cell therapy drug;
preferably, the cell therapy drug comprises an external cell therapy drug or an injection cell therapy drug.
9. A cell therapy product comprising the genetically modified mesenchymal stem cell of claim 6;
preferably, the cell therapy product comprises a cell therapy drug;
preferably, the cell therapy drug comprises an external cell therapy drug or an injection cell therapy drug.
10. Use of a cell therapy product according to claim 9 in the manufacture of a product for the treatment of paraquat poisoning.
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